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Research Article

In vitro synergism between algae and bacteria isolated from

bio-diversity hotspot for better environmental sustainability

Debapriya Roy*, Srijan Bhattacharya, Antara Biswas, Arpan Banerjee, Shinjini Ghosh and Arup Kumar

Mitra

Department of Microbiology, St. Xavier’s College (Autonomous), Kolkata.

*debapriyaroy026@gmail.com

Article Info

Abstract

Received: 22-03-2019,

Revised: 12-05-2019,

Accepted: 28-06-2019

A water sample containing algae was collected from Manipur. Microscopic

examination revealed that it contained filamentous green algae and diatoms.

Upon pour plating it gave rise to three types of bacterial colonies which were

Bacillus spp, Staphylococcus spp and Streptomyces spp. To artificially

prepared BG11 broth respective proportions of algae and bacterial cultures

were inoculated followed by constant monitoring of algal biomass after 15 and

30 days accordingly. Each of the bacteria promotes algal growth as indicated

from the in-vitro increase in the algal biomass with Bacillus spp promoting the

maximum growth. The symbiotic interaction between Bacillus spp and the

algae resulted in the increase of the algal biomass by 10.71%. So, on a detailed

study under SEM, it was found out that the algal sample procured nourishment

from the nutrients supplied by the Bacillus spp as well as the glycocalyx

adhesion of Bacillusspp with that of cellulosic algal wall. On the other hand,

Chlorophyll assay and Carotenoid assay was performed which revealed the

ability of the algae to perform photosynthesis with the help of increased

Chlorophyll b and production of Carotenoid. The measurement of Chlorophyll

a and b at 645nm and 663nm revealed the increase of Chlorophyll b

concentration by 3.05%, along with increase in Carotenoid concentration at

470nm was determined to be 0.97µg/ml. The artificial cultivation of diatoms

resulted in partial degradation of silica wall which was replenished by bacterial

conversion of dead debris of diatoms. Thus it can be concluded that the

bacterial association not only facilitate nutrient availability but also helps in

partial increase in anabolic process of the algal consortia.

Keywords:

filamentous green algae,

BG11 broth, symbiotic,

cellulosic, glycocalyx,

carotenoid, chlorophyll.

INTRODUCTION

The interaction between algae and bacteria has

always been a field of interest. The cultivation of

microalgae helps in the removal of pollutants from

wastewater, total suspended solids, total dissolved

solids etc. Microalgae turned out to be a very

promising way out for wastewater treatment where

the natural contents of the algae namely the

carbohydrate and protein part remain intact in this

clearing process. These natural contents are in turn

suitable for energy production. The high utility of

microalgae involving the wastewater treatment

along with the biofuel production settle all the

issues related to the expensive and not so

environment friendly fossil fuels. Our work thus

encompasses an integrated process uplifting the

growth of algae in aid of Bacillus spp which in turn

has multiple future prospects as mentioned above

(Raphael Slade and Ausilio Bauen, 2013).Water

sample collected from a biological hotspot was

examined. Treatment of pernicious anemia by a

special diet (Minot & Murphy, 2001).

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Debapriya Roy et al.,

The aim of this project was to study the interaction

between the algal species and the bacterial species

isolated from the water sample, when allowed to

grow under in-vitro conditions. The increase in

algal biomass and further assays revealed that all

the bacterial species were promoting algal growth

(Harold & Svec, 1966). Microscopic examination of

the water sample showed the presence of

filamentous green algae such as Spirogyra and the

average size of the algae measured under 450X

magnification was found to be 72µm.

MATERIALS AND METHODS

Pour plate technique

The pour plate technique was performed. Colonies

of different bacterial species were obtained after

incubating the petri plate overnight. Gram staining

was performed to identify the gram characteristics

of the bacterial species.

Bacterial Characterisation:

The gram staining protocols were followed

(Barthomolew and J.W et al., 1962). All the three

species exhibited purple coloration indicating that

they are gram positive. The characteristics of two

isolated colonies were gram positive rods and the

third colony was found to be gram positive cocci in

cluster.

The inoculums from incubated petri-plate were

transferred to a chrome agar medium in the form of

quadrant streaking to assume the genus of bacteria

from the color they exhibit. (J. Merlino et al., 2000).

After overnight incubation, the quadrant streaking

changed to blue colour with a white halo and green

colouration. Comparing the range of colours, they

were found to be Bacillus spp., Streptomyces spp.

Catalase is an enzyme which is produced by

microbes living in O2 rich areas, to neutralize toxic

forms of oxygen metabolites, H2O2 (F C Tenover et

al., 1994). 4-5 drops of 3% H2O2 was placed on a

clean glass slide. Small quantity of bacterial colony

was transferred into the glass slide containing H2O2

solution. Rapid effervescence showed the positive

result. The isolated bacteria may be Staphylococcus

spp.

Blood Agar is an enriched media to grow fastidious

organisms and to differentiate bacteria based on

their haemolytic properties. The isolated colony was

streaked on blood agar and incubated at 37oC for 24

hours. The colonies showed yellow colouration

surrounded by zones of clear beta haemolysis. The

isolated bacteria were confirmed to be

Staphylococcus spp.

Interaction between bacteria

Streak plate technique is used for the isolation of

pure culture of the organism (mostly bacteria) from

mixed population. T-streak method is a general

method showing possible positive and negative

interactions between the two genera of Bacteria.

The possible interactions between the

aforementioned isolated bacteria were observed

with the help of T-streaking (Beisher et al., 1995).

The possible combinations were:

a) Streptomyces spp and Bacillus spp.

b) Streptomyces spp and Staphylococcus spp.

c) Staphylococcus spp and Bacillus spp

Estimation of Algal biomass in BG11 Broth

BG11 broth is a universal medium for the

cultivation and maintenance of algal growth. This

medium supports growth of photoautotrophic algae

which requires light as a source of energy. Synthetic

nitrogen and carbon sources and also other

inorganic salts comprise this medium. The exposure

to light intensity optimizes the growth. Isolation of

algae after lyophilisation and consequent

inoculation in BG11 broth was done. (Ilavarasi et.al

2011).Then the algal suspension was kept in tissue

culture lab for 30 days under artificial light

conditions and increase in algal biomass confirmed

the observable growth.

Chlorophyll and Carotenoid assay

Spectrophotometric methods were used for the

determination of Chlorophyll a and b content in the

filamentous green algae according to (Nayek et.al

2014). An extract was prepared by filtering the

growing algae through 0.45 µm membrane filter

which were solubilized in acetone. The filters were

ground in a mortar pestle with 3-4 ml of

spectrophotometric grade acetone (90%) for 3

minutes at room temperature. The volume was

doubled with 90% acetone after grinding. It was

mixed and centrifuged for 10 minutes at 5000rpm.

The absorbance of the supernatant was determined

at appropriate wavelength

Chlorophyll a and Chlorophyll b were calculated

from the following formulas: (Nayek et al., 2014). Chl.a = 12.7(A663) – 2.69(A645)

Chl.b = 22.9(A645) – 4.68(A663)

Total Chlorophyll = 20.2(A645) + 8.02(A663)

The extracts of filamentous green algae for carotene

assay were prepared by homogenizing the algae

with 10ml of acetone (80%). The homogenized

extracts were centrifuged at 10,000rpm for 15

minutes at 4C (Minerva Lara-Flores, 2013).

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Bioscience Discovery, 10(3):134-141, July - 2019

The supernatants were separated and 0.5ml of it was

mixed with 4.5ml of acetone (80%). Then the

solution mixtures were analyzed for carotenoid

content in spectrophotometer. The O.D measured at

470nm in spectrophotometer. Carotenoid was calculated by the following

formula: (Nayek et al., 2014). C x+c = (1000A470 – 1.82Chl.a – 85.02Chl.b)/198

RESULTS AND DISCUSSION

Synergistic interaction between algae and

bacteria was observed.

12 mg of algae (Spirogyra spp) was re-suspended in

BG11 broth and bacterial suspension was made

from pure culture. Four different types of set ups

were established to analyze the synergistic

interaction between algae and bacteria. These are

the following working set ups:

Table 1: Synergistic interaction between algae and bacteria

Sl.

No

Set-up Ratio of

bacteria

and

algae

Description

1. Control - Only algal species was inoculated in 1 ml of BG11 broth.

2. Staphylococcu

s spp + Algae

1:1 1ml of Staphylococcus spp suspension inoculated in 1 ml of BG11

broth containing algae.

2:1 3ml of Staphylococcus spp suspension was inoculated in 1.5 ml of

BG11 broth containing algae.

3. Bacillus spp +

Algae

1:1 1ml of Bacillus spp suspension was inoculated in 1 ml of BG11

broth containing algae.

2:1 3ml of Bacillus spp suspension was inoculated in 1.5 ml of BG11

broth containing algae.

4. Streptomyces

spp + Algae

1:1 1ml of Streptomyces spp suspension was inoculated in 1 ml of BG11

broth containing algae.

2:1 3ml of Streptomyces spp suspension was inoculated in 1.5 ml of

BG11 broth containing algae.

These respective ratios were kept in tissue culture lab and growth was monitored for 15 and 30 days

respectively. After 30 days, 2:1 did not show any possible growth so they were eliminated.

Table 2: Ratio of bacteria and algae on biomass after 15 and 30 days

Ratio Interaction type Biomass after 15

days(mg)

Biomass after 30

days(mg)

2:1 1+A 5.5 3.2

2:1 2+A 6.0 3.1

2:1 4+A 6.0 3.1

1:1 1+A 7.9 8.4

1:1 2+A 8.4 9.3

1:1 4+A 8.4 5.4

- Control 5 .0 3.5

A – Algae

1- Staphylococcus spp

2 - Bacillus spp

4-Streptomyces spp

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Debapriya Roy et al.,

The percentage increase of the algal biomass in the

presence of Bacillus spp was found to have risen by

10.71%. Percentage increase of biomass was

observed only in the ratios 1:1. The 1:1 ratio of

algal biomass in presence of Bacillus spp showed

maximum growth after 30 days.

Analysis of algal growth by Scanning Electron

Microscopy (SEM):

Analysis by SEM revealed the interaction between

the Bacillus and the algal species. Mainly the

controlled culture and the growth of algae in the

presence of Bacillus spp was examined under SEM.

In the controlled culture (figure2: 3a,3b), where the

algae was grown invitro in BG11 broth without any

bacterial species revealed the following

observations :

Filamentous masses of Spirogyra showed varying

diameter range.Filament was relatively less thick

and the presence of conjugation bridge was

observed. The algae was observed to be in

replicating condition. Scalariform conjugation was

observed where there is association of two filaments

lined side by side partially or throughout their

length. SEM study also revealed the presence of

diatoms. Nutritional deficiency resulted in the

degradation of silica walls in diatoms. Further

analysis of interaction between algae and Bacillus

grown invitro in BG11 broth revealed the following

observations:

Filamentous green algae Spirogyra showed apical

growth when grown in interaction with Bacillus

spp. in BG11 broth.(figure: 3-5a). Most of the

Bacillus spp. was found growing on the algal

surface.(figure:3-5b). The filament was found to be

relatively thicker in comparison to the filament in

controlled culture.

Bacteria remain attached to the algal surface due to

adhesion between the glycocalyx and the algal

cellulosic wall.(figure: 3- 6a). Algae are showing

epiphytic growth in presence of bacteria. Bacteria

are providing nutrients promoting the growth of

Spirogyra and the diatoms. There is an evidence of

the regeneration of silica walls in diatoms in the

presence of bacteria which probably came from the

dead and degrading diatoms through natural cycling

(figure: 3-6b). Thus, SEM study reveals the

synergistic interaction between the algae and the

bacteria, promoting algal growth.

Table 3: Determination of Chlorophyll a, Chlorophyll b and Carotenoid content in the filamentous

green algae:

Sample Chl.a

(µg/ml)

Chl.b (µg/ml) Total Cholorophyll

(µg/ml)

Carotenoid (µg/ml)

Control 4.61 1.31 3.31 0.47

Bacillus spp + Algae

(1:1)

0.67 1.35 2.02 1.43

Thus, the results show that there is a decrease in

chlorophyll a concentration, while increase in

chlorophyll b concentration in the algal species

grown in presence of Bacillus spp, but on the other

hand there is a high increase in carotenoid

concentration. Increase in chlorophyll b

concentration was found to be 3.05% while

carotenoid concentration was found to increase by

0.97µg/ml (Banerjee and Ragsdale, 2003).

The synergistic interaction was observed between

the algal and bacterial species, which is playing a

major role in promoting algal growth. The bacteria

is providing nutrients which is utilized by the algal

species to carry out various anabolic processes

(Carlucci & Bowes, 1970). Increase in

concentration of the photosynthetic pigment

chlorophyll b and carotenoid indicates that the

algae, when grown under invitro conditions in

presence of bacteria is utilizing chlorophyll b and

carotenoid as the photosynthetic pigments to

perform photosynthesis for their survival.

Furthermore, the artificial cultivation of diatoms

resulted in partial degradation of silica wall of

diatoms which was regenerated by the bacterial

conversion of debris of diatoms (Bartholomew et

al., 1962; Kay Bidle, Farooq Azam, 2001). Thus,

there is an evidence of symbiotic association

between algae and Bacillus spp which is enabling

them to survive and replicate in invitro conditions.

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Bioscience Discovery, 10(3):134-141, July - 2019

1A 1B 1C

Figure:1 (1A, 1B, 1C) Gram staining of the bacteria observed under 450X magnification. (2A) Chrome agar

plates after streaking. Colonies showing blue coloration with white halo. (2B) Catalase test. Rapid

effervescence showing the positive result for Staphylocccus spp. (2C) Colonies exhibiting yellow coloration

surrounded by zones of clear beta haemolysis. (3A, 3B, 3C) Streaking between two genera of bacteria. (3A)

Streptomyces spp and Bacillus spp. Streptomyces spp inhibited the growth of Bacillus spp due to the possible

secretion of an antibiotic. (3B) Streptomyces spp. and Bacillus spp. Streptomyces spp inhibited the growth of

Staphylococcus spp due to the possible secretion of an antibiotic. (3C) Staphylococcus spp and Bacillus spp.

Bacillus spp inhibited the growth of Staphylococcus spp.

2A 2B 2C

3A 3B 3C

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Debapriya Roy et al.,

A – Algae

1- Staphylococcus spp

2 - Bacillus spp

4-Streptomyces spp

Figure: (3) Comparison of chlorophyll content.

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Bioscience Discovery, 10(3):134-141, July - 2019

5a 5b

Fig. 4 Graphical representation of increase in algal biomass in the presence of Bacillus spp with time.

(5a, 5b) Control culture, where the algae was grown in-vitro in BG11 broth without any bacterial species.

Figure: 6 (a,b)Interaction between algal species and Bacillus spp. studied under SEM.

6a 6b

6c 6d

6e 6f

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Debapriya Roy et al.,

(c) Filamentous apical growth of Spirogyra in presence of bacteria. (d) Magnified view of bacteria

growing on Spirogyra filament. (e) Epiphytic growth is observed. Glycocalyx interaction with algal

cellulose wall. (f) Bacterial control of silica regeneration.

REFERENCES

Banerjee R & Ragsdale SW, 2003. The many

faces of vitamin B12: catalysis by cobalamin-

dependent enzymes. Annual Review of

Biochemisty,72:209-247.

Beishir Lois, 1999. Microbiology in Practice: A

Self-Instructional Laboratory Course, Fifth Edition.

(Harper Collins: New York).

Carlucci AF & Bowes PM, 1970. Vitamin

production and utilization by phytoplankton in

mixed culture. Journal of Phycology, 6(4):393 –

400.

Merlino J, Siarakas S, Robertson GJ, Funnell GR,

Gottlieb T, Bradbury R, 1996. Evaluation of

CHROMagar Orientation for differentiation and

presumptive identification of gram-negative bacilli and

Enterococcus species. Journal of clinical Microbiology,

34(7):1788-1793.

FC Tenover, R Arbeit, G Archer, J Biddle, S

Byrne, R Goering, G Hancock, G A Hébert, B

Hill, R Hollis, 1994 February. Comparison of

traditional and molecular methods of typing isolates

of Staphylococcus aureus. Journal of Clinical

Microbiology, 32(2):407–415.

Harold H, Strainwalter A. Svec, 1966. Extraction,

separation, estimation and isolation of chlorophylls.

The chlorophylls,:21-66.

https://doi.org/10.1016/B978-1-4832-3289-

8.50008-4

JW Bartholomew, Thomas Cromwell,

and Richard Gan, 1962. Variables influencing

results, and the precise definition of steps in Gram

staining as a means of standardizing the results

obtained. Journal of Bacteriology,37: 139–155.

Kay D Bidle, Farooq Azam, 2001. Bacterial

control of silicon regeneration from diatom detritus:

significance of bacterial ectohydrolases and species

identity. Limnology and Oceanography, 46(7):

1606-1623.

Martin T Croft, Andrew D Lawrence, Evelyne

Raux Deery, Martin J warren, Alison G Smith,

2005. Algae acquire Vitamin B12 through a

symbiotic relationship with bacteria. Nature,

438(7064): 90-93.

Minerva Garcia-Chavarria and Maurilio Lara-

Flores, 2013. The use of carotenoid in aquaculture.

Research Journal of Fisheries and

Hydrobiology,8(2):38-49.

Minot GR & Murphy WP, 2001. Treatment of

pernicious anemia by a special diet. Yale Journal of

Biology and Medicine.,74(5):341-353.

Nayek Sumanta, Choudhury Imranul Haque,

Jaishee Nishika and Roy Suprakash, 2014.

Spectrophotometric Analysis of Chlorophylls and

Carotenoids from Commonly Grown Fern Species

by Using Various Extracting Solvents. Research

Journal of Chemical Sciences, 4(9):63-69.

Raphael Slade and Ausilio Bauen, 2013. Micro-

algae cultivation for biofuels: Cost, energy balance

environmental impacts and future prospects.

Biomass and Bioenergy, 53: 29-38.

Seetharam B & Alpers DH, 1982. Absorption and

transport of cobalamin (vitamin B12), Annual

Review of Nutrition, 2:343-369.

Seetharam B, Bose S & Li N, 1999. Cellular

import of cobalamin (Vitamin B12). Journal of

Nutrition,129(10):1761-1764.

Stabler SP & Allen RH, 2004. Vitamin B12

deficiency as a worldwide problem. Journal of

Nutrition,129(10):1761-1764.

Warren MJ, Raux E, Schubert HL & Escalante-

Semerena, JC, 2002: The biosynthesis of

adenosylcobalamin (Vitamin B12). Natural Product

Reports, 19: 390-412.

How to cite this article

Debapriya Roy, Srijan Bhattacharya, Antara Biswas, Arpan Banerjee, Shinjini Ghosh and Arup

Kumar Mitra, 2019. In vitro synergism between algae and bacteria isolated from bio-diversity hotspot for

better environmental sustainability. Bioscience Discovery, 10(3):134-141.